Oxidative DNA damage is caused by reactive oxygen species (ROS), which are produced both endogenously as the by-product of cellular metabolism, the inflammatory response, and ischemia-reperfusion, and exogenously through exposure to ionizing radiation and environmental carcinogens. A frequently occurring DNA base lesion produced by ROS exposure is 7,8-dihydro-8-oxoguanine (8-oxoG). 8-oxoG has the capacity to pair with bases other than cytosine during DNA replication. If left unrepaired prior to DNA replication, 8-oxoG paired with adenine in DNA could lead to the fixation of G:C to T:A transversion mutations which may result in altered gene function and promote carcinogenesis. Oxidative base damages, such as 8-oxoG, are repaired primarily by the base excision repair (BER) pathway, in which the first step is excision of the damaged base by a specific DNA glycosylase. The major mammalian enzyme for removing 8-oxoG from DNA is 8-oxoguanine-DNA glycosylase (OGG1). The importance of OGG1 in maintaining genomic integrity is underscored by a marked accumulation of 8-oxoguanine in the genome of mice lacking OGG1, and a 5-fold increased incidence of lung adenocarcinoma in these animals. In humans, the OGG1 chromosomal locus is frequently missing in lung and kidney tumors and G:C to T:A transversion mutations in the p53 gene are commonly found in lung cancer, providing additional support for the in vivo cancer suppression function of OGG1. In addition to the procarcinogenic effect of the absence of OGG1 activity, numerous OGG1 sequence variants have been reported and are associated with pathological conditions, including cancer and aging. In human populations, a frequently occurring OGG1 polymorphism results in the substitution of serine 326 for cysteine (S326C) in the C-terminus of the enzyme. Association studies have found that individuals homozygous for the S326C allele have increased incidence of lung, prostate, and orolaryngeal cancers. Significant functional changes in OGG1 caused by the S326C amino acid substitution have not been reported, thus the mechanism of carcinogenesis associated with this variant has remained unclear. We performed detailed enzymatic studies of polymorphic OGG1 under Michaelis-Menten conditions and found major functional defects in the enzyme relative to wild-type OGG1. The S326C isoform excised 8-oxoG from duplex DNA at rates at least 2-fold less than the wild-type enzyme, regardless of the base opposite 8-oxoG. AP-lyase cleavage of abasic sites and DNA damage binding affinity were also significantly decreased in polymorphic OGG1. The glycosylase activity of S326C OGG1 was not significantly stimulated by the presence of AP-endonuclease. Remarkably, gel shift studies and chemical cross-linking showed that S326C both exists in solution and binds damaged DNA as a dimer. The catalytic deficiencies and unusual DNA binding conformation of S326C OGG1 may underlie its linkage to cancer incidence.